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The emerging phenomenon of cellular heterogeneity in tissue requires single-cell resolution studies. A specific challenge for suspension-based single-cell analysis is the preservation of intact cell states when single cells are isolated from tissue contexts, in order to enable downstream analyses to extract accurate, native information. We have developed DISSECT (Disaggregation for Intracellular Signaling in Single Epithelial Cells from Tissue) coupled to mass cytometry (CyTOF: Cytometry by Time-of-Flight), an experimental approach for profiling intact signaling states of single cells from epithelial tissue specimens. We have previously applied DISSECT-CyTOF to fresh mouse intestinal samples and to Formalin-Fixed, Paraffin-Embedded (FFPE) human colorectal cancer specimens. Here, we present detailed protocols for each of these procedures, as well as a new method for applying DISSECT to cryopreserved tissue slices. We present example data for using DISSECT on a cryopreserved specimen of the human colon to profile its immune and epithelial composition. These techniques can be used for high-resolution studies for monitoring disease-related alternations in different cellular compartments using specimens stored in cryopreserved or FFPE tissue banks.
Mass cytometry is a single-cell biology technique that samples >500 cells per second, measures >35 features per cell, and is sensitive across a dynamic range of >10 relative intensity units per feature. This combination of technical assets has powered a series of recent cytomic studies where investigators used mass cytometry to measure protein and phospho-protein expression in millions of cells, characterize rare cell types in healthy and diseased tissues, and reveal novel, unexpected cells. However, these advances largely occurred in studies of blood, lymphoid tissues, and bone marrow, since the cells in these tissues are readily obtained in single-cell suspensions. This unit establishes a primer for single-cell analysis of solid tumors and tissues, and has been tested with mass cytometry. The cells obtained from these protocols can be fixed for study, cryopreserved for long-term storage, or perturbed ex vivo to dissect responses to stimuli and inhibitors. © 2017 by John Wiley & Sons, Inc.
Copyright © 2017 John Wiley & Sons, Inc.
Primary tumor organoids grown in three-dimensional culture provide an excellent platform for studying tumor progression, invasion, and drug response. However, organoid generation protocols require fresh tumor tissue, which limits organoid research and clinical use. This study investigates cellular morphology, viability, and drug response of organoids derived from frozen tissues. The results demonstrate that viable organoids can be grown from flash-frozen and thawed tissue and from bulk tissues slowly frozen in DMSO supplemented media. While the freezing process affects the basal metabolic rate of the cells, the optical metabolic imaging index correlates between organoids derived from fresh and frozen tissue and can be used to detect drug response of organoids grown from frozen tissues. The slow, DMSO frozen tissue yielded organoids with more accurate drug response than the flash frozen tissues, and thus bulk tissue should be preserved for subsequent organoid generation by slow freezing in DMSO supplemented media.
CONTEXT - Transbronchial cryobiopsy technique yields larger biopsies with enhanced quality. The benefits and safety of cryobiopsies have not been thoroughly studied in lung allografts.
OBJECTIVE - To compare size, quality, reproducibility of interpretation of rejection and complications of cryobiopsies with those of conventional biopsies from lung allografts.
DESIGN - All cryobiopsies (March 2014-January 2015) of lung allografts performed at Mayo Clinic, Rochester, and medical records were reviewed. For comparison, conventional biopsies from the same patient or, if unavailable, from a random patient, were selected. Two pathologists blinded to outcome reviewed all biopsies. Specimen volume, number of alveoli, small airways, and pulmonary vessels were counted and statistically compared.
RESULTS - Fifty-four biopsies (27 cryobiopsies) from 18 patients (11 men) were reviewed. A median of 3 (range, 2-5) and 10 (range, 6-12) specimens were obtained with cryobiopsies and conventional biopsies, respectively. Cryobiopsies were larger and contained more alveoli (P < .001, both) and small airways (P = .04). Conventional biopsies showed more fresh alveolar hemorrhage (procedural) and crush artifact/atelectasis (P < .001, both). Cryobiopsies contained more pulmonary veins and venules (P < .001). There was no significant difference between the types of biopsies with respect to the reviewers' agreement on grades of rejection. Complications were more frequent in the cryobiopsy group, though the difference was not statistically significant.
CONCLUSIONS - Cryobiopsies of lung allografts are larger and have less artifact. However, complications occur and should be considered. Three cryobiopsy specimens appear sufficient for histopathologic evaluation of lung allografts.
OBJECTIVES/HYPOTHESIS - To determine the feasibility of viable storage of head and neck squamous cell carcinoma (HNSCC) for regrowth of cells in culture.
STUDY DESIGN - Laboratory-based translational study.
METHODS - Methods for intermediate-term frozen storage of viable HNSCC were explored using small pieces of primary tumor and dissociated HNSCC cells after short-term culture. Viable cells after freezing were confirmed by adherence to tissue culture plates, cell morphology, and increased cell or colony density. Two cultures were immunostained for cytokeratin to confirm epithelial origin of viable cultured cells after freezing.
RESULTS - Six primary HNSCCs (two oral cavity, three larynx, one oropharynx) and two HNSCCs that had been passaged through a xenograft (two oral cavity) were dissociated to single cells and grown in short-term cell culture for 0 to 12 passages. After short-term culture, cells were frozen for up to 8 months, thawed, and replated. Frozen cells derived from all tumors (six primary and two xenografts) were successfully replated with cultures lasting >7 days with seven of eight tumors presenting increased colony or cell density over 1 week of growth after freezing. In total, 15 of 15 tested samples derived from six primary and two xenografted HNSCCs were viable after freezing.
CONCLUSIONS - In the current study, we show that biopreservation of primary or xenografted HNSCC using short-term cell culture is feasible. Initial short-term cell culture was required for successful storage and viability of frozen cells. These proof-of-principle studies, if more widely implemented, could improve preclinical testing of new therapies for HNSCC.
Copyright © 2013 The American Laryngological, Rhinological, and Otological Society, Inc.
STUDY DESIGN - An in vitro biomechanical study on 3-dimensional flexibility of human lumbosacral motion segments after multiple freeze-thaw cycles and cumulative testing.
OBJECTIVE - To determine the significance of multiple freeze-thaw cycles and extended testing duration on between-day and within-day variations in motion segment flexibility.
SUMMARY OF BACKGROUND DATA - Previous studies have found no significant effect of single freeze-thaw cycle on creep behavior of human spinal motion segments. Up to 3 freeze-thaw cycles were found to not affect flexibility of porcine spines and viscoelastic properties of human tendons, but more than 5 freeze-thaw cycles resulted in declined structural properties of human tendons.
METHODS - Three lumbosacral motion segments were subjected to repeated flexibility tests to determine both the effects of within-day ambient exposure and between-day multiple freeze-thaw cycles on range of motion (ROM) and neutral zone (NZ). Repeated measures analysis of variance was carried out to evaluate within-day and between-day effects at α = .05.
RESULTS - Significant between-day effects were found for intervertebral ROM and NZ in flexion-extension (FE), lateral bending (LB), and axial rotation (AR) (all P < 0.001). Post hoc analysis indicated that significant differences from test day 1 become apparent after 4 freeze-thaw cycles with cumulative testing. There were no within-day variations from repeated testing on intervertebral FE ROM (P = 0.10), LB ROM (P = 0.36), AR ROM (P = 0.46), FE NZ (P = 0.83), LB NZ (P = 0.42), and AR NZ (P = 0.72).
CONCLUSION - The flexibility of the human cadaveric lumbosacral motion segments between test days was significantly affected after repeated freeze-thaw and cumulative testing cycles. Multiple freeze-thaw cycles and cumulative testing, however, does not affect flexibility data for tests carried out within a single test day.
Engraftment and OS after umbilical CBT is highly dependent on the TNC. The contribution of the wash step to cell loss and ultimately the dose of cells available for transplant is not well described. To investigate the amount of cell loss after washing and its impact on major outcomes compared to pre-cryopreserved TNC, we analyzed data from patients prospectively enrolled on a National Heart, Lung and Blood Institute sponsored cord blood transplant study between 1999 and 2003. There were 310 patients ≤18 yr of age with malignant (N = 218) or non-malignant (N = 92) disease enrolled on this trial. Only single CBU were used. All CBU were thawed and washed using an identical process. The median TNC after thawing and washing (PTW) was 5.43 × 10(7) /kg (79% recovery of cells). The cumulative incidence of neutrophil engraftment was significantly higher in patients receiving a PTW TNC ≥2.5 × 10(7) /kg (p = 0.01). The cumulative incidence of TRM was higher among patients receiving post-thaw-and-wash TNC <2.5 × 10(7) /kg (p = 0.039). In conclusion, receiving a PTW TNC of <2.5 × 10(7) /kg resulted in worse neutrophil engraftment and increased transplant-related mortality compared to a PTW TNC of ≥2.5 × 10(7) /kg.
© 2012 John Wiley & Sons A/S.
Peptide-based mass spectrometry approaches, such as multiple reaction monitoring, provide a powerful means to measure candidate protein biomarkers in plasma. A potential confounding problem is the effect of preanalytical variables, which may affect the integrity of proteins and peptides. Although some blood proteins undergo rapid physiological proteolysis ex vivo, the stability of most plasma proteins to preanalytical variables remains largely unexplored. We applied liquid chromatography-tandem mass spectrometry shotgun proteomics and multiple reaction monitoring analyses to characterize the stability of proteins at the peptide level in plasma. We systematically evaluated the effects of delay in plasma preparation at different temperatures, multiple freeze-thaw cycles and erythocyte hemolysis on peptide and protein inventories in prospectively collected human plasma. Time course studies indicated few significant changes in peptide and protein identifications, semitryptic peptides and methionine-oxidized peptides in plasma from blood collected in EDTA plasma tubes and stored for up to a week at 4 °C or room temperature prior to plasma isolation. Similarly, few significant changes were observed in similar analyses of plasma subjected to up to 25 freeze-thaw cycles. Hemolyzed samples produced no significant differences beyond the presence of hemoglobin proteins. Finally, paired comparisons of plasma and serum samples prepared from the same patients also yielded few significant differences, except for the depletion of fibrinogen in serum. Blood proteins thus are broadly stable to preanalytical variables when analyzed at the peptide level. Collection protocols to generate plasma for multiple reaction monitoring-based analyses may have different requirements than for other analyses directed at intact proteins.
Recent observations suggest that the ubiquitin-proteasome system (UPS) contributes to the pathophysiology of myocardial ischemia-reperfusion injury. Since its regulation during cold ischemia-reperfusion is unknown, we evaluated the cardiac UPS in a model of heart transplantation in mice. Cardiac ubiquitylation rates and ubiquitin-protein conjugates increased after 3h of cold ischemia (CI) and normalized post-transplant. 20S proteasome content and proteasome peptidase activities were unchanged after CI. 4h/24h post-transplant 20S proteasome concentrations decreased and chymotryptic-like but not tryptic-like proteasome peptidase activity was inactivated. Epoxomicin sensitivity of the proteasome increased 5.7-fold during CI and normalized 4h/24h post-transplant. This was accompanied by the disappearance of a 13.5 kDa-ubiquitin-conjugate during CI that could be attenuated by addition of epoxomicin to the preservation fluid. We conclude that substrate specificity of the proteasome changes during cold ischemia and that proteasome inhibition preserves the physiological ubiquitin-protein conjugate pool during organ preservation. Reduced proteasome activity during reperfusion is caused by a decrease in proteasome content and enzyme inhibition.